BCG therapy downregulates HLA-I on malignant cells to subvert antitumor immune responses in bladder cancer
Mathieu Rouanne, … , Laurence Zitvogel, Aurélien Marabelle
Mathieu Rouanne, … , Laurence Zitvogel, Aurélien Marabelle
Published May 3, 2022
Citation Information: J Clin Invest. 2022;132(12):e145666. https://doi.org/10.1172/JCI145666.
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Research Article Immunology Oncology

BCG therapy downregulates HLA-I on malignant cells to subvert antitumor immune responses in bladder cancer

Abstract

Patients with high-risk, nonmuscle-invasive bladder cancer (NMIBC) frequently relapse after standard intravesical bacillus Calmette-Guérin (BCG) therapy and may have a dismal outcome. The mechanisms of resistance to such immunotherapy remain poorly understood. Here, using cancer cell lines, freshly resected human bladder tumors, and samples from cohorts of patients with bladder cancer before and after BCG therapy, we demonstrate 2 distinct patterns of immune subversion upon BCG relapse. In the first pattern, intracellular BCG infection of cancer cells induced a posttranscriptional downregulation of HLA-I membrane expression via inhibition of autophagy flux. Patients with HLA-I–deficient cancer cells following BCG therapy had a myeloid immunosuppressive tumor microenvironment (TME) with epithelial-mesenchymal transition (EMT) characteristics and dismal outcomes. Conversely, patients with HLA-I–proficient cancer cells after BCG therapy presented with CD8+ T cell tumor infiltrates, upregulation of inflammatory cytokines, and immune checkpoint–inhibitory molecules. The latter patients had a very favorable outcome. We surmise that HLA-I expression in bladder cancers at relapse following BCG does not result from immunoediting but rather from an immune subversion process directly induced by BCG on cancer cells, which predicts a dismal prognosis. HLA-I scoring of cancer cells by IHC staining can be easily implemented by pathologists in routine practice to stratify future treatment strategies for patients with urothelial cancer.

Authors

Mathieu Rouanne, Julien Adam, Camélia Radulescu, Diane Letourneur, Delphine Bredel, Séverine Mouraud, Anne-Gaëlle Goubet, Marion Leduc, Noah Chen, Tuan Zea Tan, Nicolas Signolle, Amélie Bigorgne, Michael Dussiot, Lambros Tselikas, Sandrine Susini, François-Xavier Danlos, Anna K. Schneider, Roman Chabanon, Sophie Vacher, Ivan Bièche, Thierry Lebret, Yves Allory, Jean-Charles Soria, Nicholas Arpaia, Guido Kroemer, Oliver Kepp, Jean Paul Thiery, Laurence Zitvogel, Aurélien Marabelle

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Figure 1

BCG induces HLA-I downregulation on cancer cells.

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BCG induces HLA-I downregulation on cancer cells. (A) Cell suspensions f...
(A) Cell suspensions from freshly dissociated human bladder tumors were cultured in triplicate in complete medium (untreated), stimulated with IFN-γ, or coincubated with BCG. (B) Gating strategy adopted to detect CD45+ immune cells and CD45–EpCAM+ cancer cells by flow cytometric analysis. (C) Proportions of HLA-I+CD45–EpCAM+ cells among live tumor cells following ex vivo BCG or IFN-γ stimulation of fresh human tumors (n = 12; 1-way ANOVA with Tukey’s post test). (D) MFI of HLA-I on live CD45–EpCAM+ cancer cells upon ex vivo BCG or IFN-γ exposure (n = 12 paired samples; 1-way ANOVA with Tukey’s post test). (E) High-grade (n = 4) and low-grade (n = 2) human bladder cancer cell lines were cultured in vitro in triplicate under 3 conditions: untreated, stimulated with IFN-γ, or coincubated with BCG. (F) Histogram showing HLA-I downregulation in a subset of cancer cells upon BCG exposure. (G) HLA-I MFI by flow cytometry 24 hours after in vitro BCG or IFN-γ exposure (n = 3 conditions per cell line; 1-way ANOVA with Tukey’s post test). (H) HLA-I+ and HLA-I– cancer cells were sorted after 24 hours of coincubation with BCG. HLA-I+ and HLA-I– cancer cells were cultured in BCG-free medium for 6 additional days, followed by flow cytometric analysis on day 7 to measure HLA-I expression on the cancer cells. (I) Sustained HLA-I MFI of cancer cells after 6 days in BCG-free medium. Left inset: Illustrative MFI from HLA-I+ (blue) and HLA-I– (red). Graph shows cumulative data points of HLA-I MFI from 5 distinct cancer cells lines (n = 5 independent experiments using RT4, 5637, HT1376, TCCSUP, and UM-UC3 cancer cell lines; unpaired, 2-tailed t test). (J) HLA-I+ and HLA-I– cancer cells were restimulated with IFN-γ for 24 hours after 3 days of culturing in BCG-free medium (1-way ANOVA with Tukey’s post test). All data are presented as the mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001.